Mechanistic studies of FosB: a divalent-metal-dependent bacillithiol-S-transferase that mediates fosfomycin resistance in Staphylococcus aureus

Abstract

FosB is a divalent-metal-dependent thiol-S-transferase implicated in fosfomycin resistance among many pathogenic Gram-positive bacteria. In the present paper, we describe detailed kinetic studies of FosB from Staphylococcus aureus (SaFosB) that confirm that bacillithiol (BSH) is its preferred physiological thiol substrate. SaFosB is the first to be characterized among a new class of enzyme (bacillithiol-S-transferases), which, unlike glutathione transferases, are distributed among many low-G + C Gram-positive bacteria that use BSH instead of glutathione as their major low-molecular-mass thiol. The Km values for BSH and fosfomycin are 4.2 and 17.8 mM respectively. Substrate specificity assays revealed that the thiol and amino groups of BSH are essential for activity, whereas malate is important for SaFosB recognition and catalytic efficiency. Metal activity assays indicated that Mn2+ and Mg2+ are likely to be the relevant cofactors under physiological conditions. The serine analogue of BSH (BOH) is an effective competitive inhibitor of SaFosB with respect to BSH, but uncompetitive with respect to fosfomycin. Coupled with NMR characterization of the reaction product (BS– fosfomycin), this demonstrates that the SaFosB-catalysed reaction pathway involves a compulsory ordered binding mechanism with fosfomycin binding first followed by BSH which then attacks the more sterically hindered C-1 carbon of the fosfomycin epoxide. Disruption of BSH biosynthesis in S. aureus increases sensitivity to fosfomycin. Together, these results indicate that SaFosB is a divalent-metal-dependent bacillithiol-S-transferase that confers fosfomycin resistance on S. aureus.

Importance of Bacillithiol in the Oxidative Stress Response of Staphylococcus aureus

“…In previous studies, FosB was shown to mediate fosfomycin resistance in S. aureus through the use of BSH to disrupt the epoxide ring of fosfomycin (18,19,38). We investigated here the role of BSH in fosfomycin resistance mediated by FosB in a wide variety of methicillin-sensitive and methicillin-resistant S. aureus strains, including clinical isolates.…”

“…Intracellular reduced thiols (BSH, Cys, and coenzyme A [CoA] sulfhydrate [CoASH]) were derivatized, in triplicate, with monobromobimane (mBBr). Samples for disulfide analysis were first treated with Nethylmaleimide (NEM), followed by dithiothreitol (DTT) reduction and labeling with mBBr of reduced thiols and oxidized disulfides (BSSB and cystine), and were analyzed by fluorescent high-pressure liquid chromatography (HPLC), as previously described (18,25). However, it is not possible to accurately quantify CoA disulfide because the DTT treatment used in the disulfide analysis also cleaves thioesters such as acetyl-CoA into CoA, resulting in an overestimate of CoA disulfide (26).…”

“…In vitro, FosB purified from S. aureus has been reported to function as a BSH-S-transferase (BST) to inactivate fosfomycin by opening up the epoxide ring of fosfomycin through the conjugation of BSH to the compound and, hence, inactivating the drug (17)(18)(19). While factors such as importers (e.g., GlpT [16] and UhpT [20]) have been connected to fosfomycin resistance, their exact contribution to resistance in S. aureus depends on transport of the drug rather than direct inactivation of FosB.…”

Role of the CpxAR Two-Component Signal Transduction System in Control of Fosfomycin Resistance and Carbon Substrate Uptake

“…Mechanisms for fosfomycin resistance are encoded on plasmids and the chromosome. Resistance to fosfomycin is conferred by the production of the plasmid-encoded enzymes FosA, FosB, FosC, and FosX, which leads to inactivation of fosfomycin (13)(14)(15)(16). Chromosomal mutations that lead to the expression of MurA variants that have impaired fosfomycin binding or result in overexpression of MurA also confer resistance to fosfomycin.…”

Regulation of Bacillus subtilis bacillithiol biosynthesis operons by Spx

“…Note that fosfomycin resistance provides a semiquantitative measure of in vivo BSH levels since resistance is largely dependent on the BSH-dependent thiol-transferase FosB. Since the K m of FosB for BSH is in the millimolar range (higher than in vivo levels during growth; Roberts et al, 2013), FosB activity, and therefore fosfomycin resistance, is very sensitive to even small changes in BSH levels (Gaballa et al, 2010;Parsonage et al, 2010). Since pMUTIN disruptants are generally polar on downstream genes (Fig.…”